Development of the High-Affinity Carborane-Based Cannabinoid Receptor Type 2 PET Ligand [18F]LUZ5-d8

The development of cannabinoid receptor type 2 (CB2R) radioligands for positron emission tomography (PET) imaging was intensively explored. To overcome the low metabolic stability and simultaneously increase the binding affinity of known CB2R radioligands, a carborane moiety was used as a bioisostere. Here we report the synthesis and characterization of carborane-based 1,8-naphthyridinones and thiazoles as novel CB2R ligands. All tested compounds showed low nanomolar CB2R affinity, with (Z)-N-[3-(4-fluorobutyl)-4,5-dimethylthiazole-2(3H)-ylidene]-(1,7-dicarba-closo-dodecaboranyl)-carboxamide (LUZ5) exhibiting the highest affinity (0.8 nM). Compound [18F]LUZ5-d8 was obtained with an automated radiosynthesizer in high radiochemical yield and purity. In vivo evaluation revealed the improved metabolic stability of [18F]LUZ5-d8 compared to that of [18F]JHU94620. PET experiments in rats revealed high uptake in spleen and low uptake in brain. Thus, the introduction of a carborane moiety is an appropriate tool for modifying literature-known CB2R ligands and gaining access to a new class of high-affinity CB2R ligands, while the in vivo pharmacology still needs to be addressed.


■ INTRODUCTION
The endocannabinoid system (ECS), named after the cannabis plant, 1 has become the focus of medicinal research, due to its involvement in the modulation of various physiological and pathological processes. 1,2−7 The endocannabinoid system is defined as the entirety of the cannabinoid receptors, the endogenous lipids, and the enzymes responsible for the synthesis and degradation thereof 8 and is still being continuously studied.Other receptors like GPR18 and GPR55 have also been shown to be modulated by cannabinoids, despite sharing a low degree of sequence homology with the CB 1 R and CB 2 R. 9,10 The CB 1 R and CB 2 R are part of the family of G proteincoupled receptors (GPCRs) and exhibit a degree of similarity of 44% for amino acids across the entire protein and of 68% regarding the transmembrane domains. 4,11The binding pockets of the CB 1 R and CB 2 R differ partly from each other. 12Recently, the antagonist and agonist bonding of the CB 2 R in the complex with corresponding ligands was revealed through crystal structures. 11,12With the knowledge of similarities and differences between the two receptors, especially the constitution and conformation of their binding pockets, the development of selective and high-affinity ligands is being actively pursued.
−17 The CB 2 R is mainly associated with the immune system and predominantly expressed among others in spleen, tonsils, and thymus. 18,19In the brain, the CB 2 R is overexpressed under pathological conditions like inflammation, 20 neurodegenerative diseases, such as Alzheimer's, Huntington's, and Parkinson's 21 diseases, or cancer. 20The CB 2 R is also connected to rheumatoid arthritis or arteriosclerosis. 19The activation of the CB 2 R can lead to beneficial effects, like causing apoptosis of cancer cells. 20The diagnosis and treatment of the aforementioned pathological conditions could be achieved by the use of selective CB 2 R ligands.The development of such compounds is ongoing and challenging, because of the strong requirements.The affinity must be high [(sub)-nanomolar range], due to the low expression density of the CB 2 R, and the selectivity must be high (1000-fold higher than for the CB 1 R) to avoid the undesired psychedelic effects of the CB 1 R. Other important characteristics are the metabolic stability, bioavailability, and ability to penetrate membranes or the blood−brain barrier (BBB) (LogP range of 1−3.5). 15,22To date, no selective CB 2 R ligand is approved for routine clinical use.To gain more insights into the involvement of the CB 2 R in physiological and pathological processes and to take action for their treatment, the theranostic approach is greatly important.A suitable tool for diagnosis is positron emission tomography (PET), a non-invasive imaging method, with which biochemical features in the body can be monitored. 23In addition to the already mentioned requirements for suitable CB 2 R ligands, it is necessary that these compounds can be radiolabeled with a suitable radionuclide for PET.The most frequently used radioisotopes for PET are 11 C (half-life of 20.4 min) and 18 F (half-life of 109.8 min). 24For the usage of PET tracers, it is important that no radiometabolites are present in the brain and that the level of interaction with plasma proteins is as low as possible. 15Evens et al. developed the oxoquinoline-based compound [ 11 C]NE40 ([ 11 C]1) 25 (Figure 1), a CB 2 R radiotracer tested in patients with Alzheimer's disease (AD).Whereas promising results have been obtained in healthy humans, the expected better binding of the tracer was not seen in the AD patients, which could possibly be related to the unfavorable biochemical properties of the radioligand. 26−29 Spinelli et al. published a review in 2017 about structure−affinity and structure−activity relationships of several CB 2 R scaffolds and identified their influence and the importance of different functional groups within the structures on their suitability as diagnostic and therapeutic CB 2 R ligands. 27A selection of known radiolabeled CB 2 R ligands is shown in Figure 1.The thiazole-based [ 11 C]A-836339 ([ 11 C]2) is an auspicious lead structure.The non-radiolabeled compound was synthesized by Dart et al., 30 further investigated by Yao et al., 31 and radiolabeled by Horti et al. 32 Extensive medicinal chemistry studies performed by us [[ 18 F]JHU94620 ([ 18 F]3)] 28,33 and Caillé[[ 18 F]FC0(3)24, in the literature as either FC024 or FC0324 ([ 18 F]4)] 24,34−36 led to the development of 18 F-labeled analogues of [ 11 C]2.While [ 18 F]3 is a low-nanomolar affinity and selective CB 2 R PET radioligand, the presence of large fractions of radiometabolites in mouse brain limits its application as a PET tracer. 28The preclinical evaluation of [ 18 F]4 in Rhesus monkeys also showed a high in vivo metabolism. 35−36 Journal of Medicinal Chemistry of a PET radioligand for CB 2 R imaging. 27,37,38  F]6). 38Recently, Modemann et al. reported an indolebased 18 F-labeled radioligand ([ 18 F]9). 39The 1,8-naphthyridine-2-one, as another lead structure, was systematically and manifold modified in the past several years. 20,27We recently reported [ 18 F]LU14 ([ 18 F]7), 40 a stereochemically pure 1,8naphthyridine-2-one radioligand, and [ 18 F]LU13 ([ 18 F]8), 41 a promising high-affinity radiotracer for targeting CB 2 R overexpression in neurological disease.
−45 No CB 2 R PET tracer is currently available for routine clinical usage.−50 The icosahedral dicarba-closo-dodecaboranes(12) are cluster compounds consisting of two carbon, 10 boron, and 12 hydrogen atoms and can be divided into ortho (o)-, meta (m)-, and para (p)-carborane (Figure 3). 51The three isomers can be transformed into each other (Figure 3).They differ not only in size and volume [core volumes of 11.79 Å 3 (ortho), 11.72 Å 3 (meta), and 11.71 Å 3 (para); van der Waals volumes (V vdW ) based on crystal structures of 148 Å 3 (ortho), 143 Å 3 (meta), and 141 Å 3 (para)] 51 but also in their reactivity.In particular, the reactivity toward Lewis bases is highest for o-carborane and lowest for p-carborane.The o-carborane is most prone to undergo a deboronation reaction that leads to the nido cluster (Figure 3). 51,52ue to the delocalization of σ-bonding electrons of the cluster, carboranes are often termed three-dimensional σ-   51,65,66 aromatic compounds and compared to benzene (π aromaticity); 52 however, the average V vdW of carboranes (144 Å 3 ) is comparable to that of adamantane (136 Å 3 ). 51Considering the chemical difference between the acidic CH units and the hydridic BH units, orthogonal regioselective substitution reactions at both moieties are possible 51 and offer a very powerful tool for modifying and adjusting the properties. 46−61 Here, the hydrophobicity can facilitate the passage of cellular membranes and the BBB and is therefore especially important for monitoring effects in the brain. 46−50 Furthermore, the carborane cluster can interact in a noncovalent way with the chosen target, e.g., by forming dihydrogen bonds. 46n the past several decades, carborane-based drug mimetics have attracted much attention. 46Endo et al. synthesized a carborane-based estradiol analogue 62 [BE120 (CB-1) (Figure 4)].Scholz et al. reported asborin 63 [CB-2 (Figure 4)], the carborane analogue of aspirin (acetylsalicylic acid).Vaźquez et al. published a rimonabant-derived CB 1 R antagonist 64 [CB-3 (Figure 4)] bearing a carborane moiety.So far, no CB 2 R ligand bearing a carboranyl moiety has been published, neither as a drug nor as a radiolabeled PET tracer.
We here report the synthesis and investigation of the first carborane-based CB 2 R ligands, which were obtained by replacing either the tetramethylcyclopropyl unit in JHU94620 28 or the adamantyl residue in LU13 41 with o-, m-, or p-carborane.All new closo-carborane-containing CB 2 R ligands described herein were evaluated by in vitro metabolism studies using liver microsomes.To gain the first insights into the in vivo behavior of such carborane compounds, we designed a radiosynthesis for our most CB 2 R potent derivative LUZ5 [compound 16].While a great number of aliphatic radiofluorinated PET candidates suffer from fast in vivo metabolism yielding free [ 18 F]fluoride, 67,68 the use of deuterated alkyls was shown to considerably improve the in vivo stability. 69Thus, a precursor bearing a fully deuterated Nbutyl chain was synthesized and radiofluorinated in the presence of K[ 18 F]F-K 2.2.2 to give [ 18 F]LUZ5-d 8 (Figure 5).The in vivo behavior of [ 18 F]LUZ5-d 8 was evaluated in metabolic and PET studies in vivo in healthy female CD-1 mice and healthy male Wistar rats, which are the first preliminary steps in the development of a theranostic agent.

■ RESULTS AND DISCUSSION
Synthesis.The synthesis of the starting material 4fluorobutyl-substituted 1,8-naphthyridin-2-one-3-carboxylic acid 4 was performed in three steps starting from 2aminopyridine-3-carbaldehyde (1) as described by Lucchesi  et al. and Sircar et al. 20,70 In brief, aldehyde 1 was reacted in a Knoevenagel condensation with diethylmalonate and piperidine as the base.A nucleophilic substitution at the NH group of ester 2 introduced the 4-fluorobutyl chain followed by a basic ester hydrolysis of 3 with LiOH•H 2 O to give 4 (Scheme 1). 71or the next step (Scheme 1, h), the corresponding carboranyl amines were required.1-Amino-1,2-dicarba-closododecaborane (6 o ) and 1-amino-1,7-dicarba-closo-dodecaborane (6 m ) were synthesized as published by Nie et al. 72 starting from o-(5 o ) or m-carborane (5 m ) (Scheme 1).However, 1-amino-1,12-dicarba-closo-dodecaborane (6 p ) could not be obtained by the same procedure or by varying the reaction time and temperature.Carboranyl amine 6 p could finally be obtained by following the synthetic approach of Tsuji et al. 73 via a multistep synthesis starting from the monosubstituted pcarborane-1-carboxylic acid 7 p , which is first chlorinated in situ followed by nucleophilic attack of trimethylsilyl azide at the carboxylic acid chloride and a Curtius rearrangement.The resulting isocyanate is reacted with tert-butanol to form the corresponding carbamate.Finally, the tert-butyloxycarbonyl (Boc) group was removed with trifluoroacetic acid (TFA) at  room temperature (rt) to give compound 6 p in 8% overall yield.Compound 6 p was used without further purifications for the next synthetic step.
The synthesis of naphthyridine-based target compounds 9− 11 (Scheme 1) was adapted from Vaźquez et al. 64 Carboxylic acid 4 was first converted to the corresponding acid chloride that was further reacted with the respective carboranyl amines 6 o , 6 m , and 6 p in a microwave reaction at 150 °C to give amides 9−11, respectively, which were isolated in 42−50% yield by column chromatography.The three compounds were characterized by one-dimensional ( 1 H, 11 B{ 1 H}, and 13 C{ 1 H}) and two-dimensional (COSY, HSQC, and HMBC) NMR spec-   troscopy and ESI-HRMS.Single crystals of o-carborane derivative 9 (Figure 6, left) and m-carborane analogue 10 (Figure 6, right) suitable for X-ray structure determination could be obtained from CDCl 3 (9) or n-hexane/EtOAc/EtOH (10 and 11) (additional information is available in Table S1; the molecular structure of 11 is shown in Figure S82).9 was deboronated with NaF in EtOH/H 2 O [1:1 (v/v)] in a microwave reaction, 74,75 yielding racemic nido-carborate(−1) derivative 12 with sodium as the counterion in 79% yield.
One of the (major) side products in the synthesis of LUZ5 could be identified as the N3-unsubstitued thiazole-carborane acid amide (SP1) (Figures S39, S40, and S58) as shown by 1 H and 11 B{ 1 H} NMR spectroscopy.
Stability Tests.A high chemical and metabolic stability of the compounds is required for biological evaluation and application in vivo.The method of choice for evaluating the chemical stability was 1 H and 11 B{ 1 H} NMR spectroscopy, with the latter being particularly suitable for recognizing deboronation.Since the in vitro binding assays are performed in aqueous media, the stability tests should be performed under similar conditions.However, due to the high hydrophobicity of the carborane moieties, target compounds A general stability trend was observed with o-carborane derivatives having the lowest and p-carborane derivatives having the highest stability.This observation was in agreement with our expectations, because o-carboranes have the highest chemical reactivity of the three isomers and are most prone to undergo deboronation reactions, especially in aqueous solutions or in the presence of strong bases. 51hiazole-based compounds 15 and LUZ5 proved to be more stable than naphthyridine isomers 9 and 10, respectively.For compound 9, additional signals were visible already after 2 h in the 1 H or 11 B{ 1 H} NMR spectra, indicating that a deboronation to nido-cluster derivative 12 had started.Thus, the stability of 9 is too low considering the time necessary to perform the binding affinity assay, which is 90 min.The other target compounds (10, 11, 15

Journal of Medicinal Chemistry
For all target compounds, K i values in the nanomolar range could be determined (Table 1

and Figures S84−S87).
Naphthyridine-based compounds 9−11 showed an increasing affinity of the o-to p-carborane derivatives toward the CB 2 R. Compound 11 (K i = 3.9 nM) showed an affinity (∼10-fold) considerably higher than those of the other two compounds.Despite the low nanomolar range, the affinity of compound 11 is lower than that of reference compound LU13 (Figure 1).Due to the low stability of 9, the binding affinity determination might be influenced by the presence of small amounts of the nido derivative 12 that is currently under evaluation.
From the thiazole-based compounds, 17 and LUZ5 showed remarkably high affinities of 1.3 and 0.8 nM, respectively, for the CB 2 R, surpassing the affinity of lead compound JHU94620 (Figure 1).Moreover, compound LUZ5 proved to be selective for the CB 2 R with a CB 1 R binding affinity of >10 μM (Figure S88).Compounds 9−11, 15, and 17 at concentrations of up to 1 μM did not displace the CB 1 R-specific radioligand [ 3 H]SR141716A (Figure S96), and thus, we conclude that these compounds bind with only negligible affinity for the CB 1 R (Figure S89).
The finding that the thiazole-based compounds display a binding affinity higher than those of the respective naphthyridine-based ones is in agreement with the binding energies of the best docked positions (Figure S90).Therefore, for 9 with the lowest binding affinity, the binding energy is the highest (−4.In Vitro Metabolism Studies.In vitro metabolism studies were performed for target compounds 9−11, 15, LUZ5, and 17.Each of the compounds was incubated with mouse liver microsomes (MLMs) in phosphate-buffered saline (PBS) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) for 30 and 60 min at 37 °C, according to a literature protocol. 81,82After the addition of cold acetonitrile, the obtained supernatants were analyzed by HPLC-UV-MS (exemplified chromatogram and spectrum in Figures S91 and  S92).The correct performance of the experimental setup was confirmed by complete conversion of testosterone under similar conditions.In addition, negative control samples without NADPH or without NADPH and MLM were prepared and analyzed for each target compound.
For each of the compounds investigated, no significant formation of metabolites was detected under the used conditions.This leads to the assumption that cytochrome P450-catalyzed functionalization reactions (phase I), such as oxidation, are not involved in the metabolism of this class of compounds in MLMs.However, the use of human liver microsomes (HLMs) instead of MLMs might provide a different picture, as species differences are described well in the literature 83,84 and were already reported for CB 2 R ligands by Aly et al. 85 However, as discussed in the section In Vivo Metabolism, radiometabolites were detected in plasma from rodents after administration of [ 18 F]LUZ5-d 8 .These findings suggest further in vitro investigations with regard to conjugation reactions (phase II), to understand the complexity of the processes taking place in vivo.
Radiochemistry.On the basis of the binding affinity and stability tests, LUZ5 was selected as the most promising candidate for developing a CB 2 R radioligand for further biological evaluations.
The synthesis of precursor 19 was performed in two steps starting from 2-amino-4,5-dimethylthiazole (13) and mcarboranyl acid chloride (8 m ) (Scheme 3).The synthesis of intermediate 18 was analogous to the syntheses of 15, LUZ5, and 17 (Scheme 2). 79For alkylation, the N3 position of 18 was deprotonated with NaH and reacted with 1,4-dibromobutane-d 8 to give 19 (Scheme 3). 28The fully deuterated chain was introduced to confer metabolic stability. 40,41,69Brominated precursor 19 was purified by HPLC and then radiofluorinated via nucleophilic substitution under thermal conditions to give The reaction conditions for the radiosynthesis of [ 18 F]LUZ5-d 8 were investigated by using various amounts of 2.2.2-cryptand (K 2.2.2 ) (13−40 μmol), K 2 CO 3 (7.5−15μmol), and precursor 19 (2.3−4.5 μmol) in CH 3 CN as the solvent under thermal conditions.Under each of the tested reaction conditions, [ 18 F]LUZ5-d 8 was detected in the range between 25% and 50% (radio-HPLC, not isolated).On the basis of these preliminary results, an automated radiosynthesis for [ 18 F]LUZ5-d 8 was developed on an Elysia-Raytest radiosynthesizer.The product was purified by HPLC (Figure 8A) followed by trapping the radiotracer [ 18 F]LUZ5-d 8 on a reverse phase (RP) cartridge and elution with EtOH.For biological experiments, the solvent was evaporated under a stream of nitrogen at 70 °C.The final product was formulated in sterile isotonic saline up to a final concentration of <10% EtOH.The overall synthesis time was ∼85 min.[ 18 F]LUZ5-d 8 was obtained in 5−8% radiochemical yield (decay-corrected from the end of bombardment), high radiochemical purity [>99% (Figure 8B)], and high molar activity in the range of 170−190 GBq/μmol at the end of the synthesis (n = 5).The identity of the final product [ 18 F]LUZ5-d 8 was confirmed with analytical HPLC by co-injection with the corresponding reference compound [co-injection (Figure 8B); UV profile of the isolated radiotracer (Figure S93)].No decomposition of the formulated product was observed within 24 h at room temperature.A logD 7.4 of 3.0 was experimentally determined for [ 18 F]LUZ5-d 8 by the shake-flask method (n = 3), which is well within the range of 1−3.5 recommended for braintargeting compounds. 22,86n Vivo Metabolism.In vivo metabolism studies of [ 18 F]LUZ5-d 8 were carried out in healthy female CD-1 mice and healthy male Wistar rats.Samples of blood plasma, brain homogenates, spleen homogenates, and urine were obtained 30 min post-injection (p.i.), and organic solvent denaturation was performed to remove proteins.The extraction was carried out in mice samples by using either MeOH/H 2 O [9: , whereby an extraction efficiency of >94% was achieved using the former (n = 3).As a result, these conditions were also used to investigate the metabolic stability of [ 18 F]LUZ5-d 8 in rats (n = 2).
In all investigated rat samples, larger amounts of an intact radiotracer were found compared to mice (Figure 9).For both mammals, significant amounts of radiometabolites were determined in blood plasma, with mean values of 19.6 ± 9% intact radiotracer in mice and 41.8 ± 0.6% in rats.Compared to the organic analogues [ 18 F]JHU94620 and [ 18 F]FC0(3)24 (Figure 1), an average larger amount of intact [ 18 F]LUZ5-d 8 could be found in plasma of mice (19.6 ± 9% compared to 7% for [ 18 F]JHU94620) 28 and rats [41.8 ± 0.6% compared to 25% for [ 18 F]FC0(3)24], 24 respectively.Also in the brain samples, a much larger amount of intact radioligand was found in both mouse (81.5 ± 1.5%) and rat samples (100%) when compared to that of [ 18 F]JHU94620 [36%, data not reported for [ 18 F]FC0(3)24]. 24,28In the spleen samples, 94 ± 5% of intact tracer for mice and 100% of intact tracer for rats were found (Figure 9).The high metabolic stability can be explained by the presence of the carborane moiety and the implementation of the deuterated substituent at the N1 position of the molecule.Both structural changes might be influencing the stability toward enzymes in vivo and therefore increase the metabolic stability.In urine, no intact radiotracer could be detected.
Assessment of the Biodistribution of [ 18 F]LUZ5-d 8 in Rodents.The biodistribution of [ 18 F]LUZ5-d 8 over time was evaluated by dynamic PET imaging under control and blocking conditions using CB 2 R agonist GW405833 (Figure S96) in healthy male Wistar rats (Figure 10 and Table 2) and in a healthy female CD-1 mouse (Figure S95).
In rats, the analysis of the time−activity curves (TACs) of the left ventricle revealed a comparable radioactivity concentration over time in the blood pool AUC 0−30 min (area under the curve between 0 and 30 min p.i.) with comparable time-to-peak (TTP) and TAC peak values (TPV) between the control and blocking group.The spleen uptake of [ 18 F]LUZ5d 8 with a TPV of 9.5 ± 2.6, 2.1 ± 0.3 min post-injection, was high and comparable to the uptake after blocking.This was also reflected by the normalized spleen SUV (standardized uptake value) to the blood pool activity with a mean SUVr (SUV ratio of x to reference) between 10 and 22 (control) and 10 and 16 (blocking) from 2 to 30 min p.i. of the radiotracer.Thus, a displaceable uptake of [ 18 F]LUZ5-d 8 into the spleen under these conditions could not be shown in vivo.The radiotracer was mainly hepatobiliary excreted as shown by the high AUC 0−30 min values of the liver, jejunum, and stomach compared to kidney and the rather low accumulated bladder radioactivity of 0.3 ± 0.1% of the injected activity after 60 min p.i. in the control group (Figure S94).The hepatobiliary excretion was delayed by the blocking compound, which most likely caused the higher renal radioactivity accumulation as shown by the TTP and AUC 0−30 min values and the decreased radioactivity concentration in the small intestine.The dynamic PET recordings were limited by the field of view of the device, so altered bladder activity under blocking conditions could unfortunately not be measured, as determination of blood pool activity was prioritized.A negligible radioactivity uptake could be observed in lung, muscle, and bone.Furthermore, the brain uptake of [ 18 F]LUZ5-d 8 in the initial distribution phase was rather low and followed by a fast washout in rats as shown in panels C and D of Figure 10.
Compared to rats, in an exemplary CD-1 mouse (Figure S95) the spleen uptake was lower, with a TPV of 1.2 after 7.5 min p.i.A high hepatobiliary excretion in mouse was found, as well, whereas the activity in bladder after 30 min was 1.9% of the injected radioactivity and therefore 10 times higher than that in rats, which could be in part explained by speciesdependent radiometabolite formation.In mouse, the brain uptake as well as uptake in muscle and bone was low and comparable to that in rats.Taken together, the uptake of [ 18 F]LUZ5-d 8 in the CB 2 Rrich tissue spleen was higher in rats than in mice but could not be blocked by the CB 2 R agonist GW405833.With the exception of the excretion pathway (mainly hepatobiliary), hints of an increased off-target uptake of the radioligand in tissues with a low CB 2 R density could not be found in both species.However, the brain uptake is rather low and a slow defluorination of [ 18 F]LUZ5-d 8 can be assumed due to the low level of accumulation of radioactivity in the bone.

■ CONCLUSIONS
In this study, a new class consisting of seven carborane-based CB 2 R ligands has been developed and evaluated.The lead structures of target compounds 9−11, 15, 16 (LUZ5), and 17, based on 1,8-naphthyridinone or thiazole scaffolds, were modified by introducing o-, m-, and p-carborane moieties.The nido-compound 12 was generated via deboronation of 9. Compounds 11, 15, and 17 showed good chemical stability in aqueous DMSO-d 6 and low nanomolar affinities (K i = 3.3, 3.9, and 1.3 nM, respectively).The binding affinity of the thiazole derivatives surpassed that of the naphthyridine derivatives.In this study, we identified LUZ5 with subnanomolar affinity exceeding the reference compound JHU94620.Encouraged by these results, we developed an automated radiofluorination for [ 18 F]LUZ5-d 8 .The in vivo metabolic stability of [ 18 F]LUZ5-d 8 bearing a deuterated butyl chain was investigated in rodents.While in mouse plasma only a small fraction of intact tracer was found 30 min p.i., the percentage of intact radioligand in mouse brain and spleen was high (81.5 ± 1.5% and 94 ± 5%, respectively) and superior to that of [ 18 F]JHU94620.The metabolic stability of [ 18 F]LUZ5-d 8 in rats exceeded that in mice.Dynamic PET scans in rats with and without blocking agent GW405833 revealed a fast uptake in spleen and liver, a fast hepatic clearance, and nondisplaceable binding in spleen.The uptake of radiotracer in brain was low.The development of [ 18 F]LUZ5-d 8 gave access to a new class of ligands for CB 2 R bearing a carborane moiety as a bioisostere for an adamantyl or tetramethylcyclopropyl group, which could alter and improve the properties of existing ligands and help to overcome the metabolic stability problems of the known CB 2 R ligands.For the use of such ligands as PET tracers targeting brain, further structural adjustments have to be considered.The foundation for further research toward diagnostic agents for theranostic application has been laid with this preliminary evaluation of an 18 F-labeled radiotracer.
■ EXPERIMENTAL SECTION General Information.All reactions involving carboranes were carried out under a nitrogen atmosphere using the Schlenk technique.Anhydrous DCM, Et 2 O, n-hexane, and toluene were dried with the MB SPS-800 solvent purification system (MBRAUN, M.Braun Inertgas-Systeme GmbH, Garching, Germany).Acetonitrile and DMF were dried over calcium hydride and distilled.Dry solvents were stored over molecular sieves (4 or 5 Å).Benzyl azide was prepared according to the literature. 87Dicarba-closo-dodecaborane-1carboxylic acids (7 o , 7 m , and 7 p ) can be prepared according to the literature. 76,77Dicarba-closo-dodecaborane-1-carboxylic acid chlorides (8 o , 8 m , and 8 p ) can be synthesized as described by Kasar et al. 78 1,2and 1,12-Dicarba-closo-dodecaborane-1-amine 6 o and 6 p can be prepared as described by Nie et al. 72 and Tsuji et al. 73 The synthesis of 1,7-dicarba-closo-dodecaborane-1-amine 6 m was adapted from Nie et al. 72 Compound 4 can be prepared according to a previously published protocol. 20,41,70,71Compound 14 can be synthesized as published by Moldovan et al. 28 All other solvents and chemicals were commercially available and used without further purification.Microwave reactions were performed by using an Initiator+ microwave from Biotage (Uppsala, Sweden).Reaction progress and purification of products were monitored by thin-layer chromatography (TLC) using precoated silica gel 60 F 254 Alumgram plates (Xtra SIL G) from Macherey-Nagel (Duren, Germany).Parts of TLC plates containing carboranes were stained with a 5−10% PdCl 2 solution in methanol.Chromatography was performed in air, with silica gel (60 Å, 0.035−0.070mm particle diameter) or in an automated fashion with an Isolera-4 and ELSD 1080 (Biotage) with commercially available solvents.

(Z)-N-[3-(4-Fluorobutyl)-4,5-dimethylthiazole-2(3H)-ylidene]-(1,2dicarba-closo-dodecaboranyl)-carboxamide
Binding Affinity.The in vitro binding affinity assays were performed with membrane homogenates from Chinese hamster ovary (CHO) cells stably transfected with the human CB 2 R according to a previously published protocol. 95The screening for CB 1 R binding affinity determination was performed with membrane homogenates from CHO cells stably transfected with the human CB 1 R as described previously. 96n Vitro Metabolism Studies.For in vitro evaluation of metabolites, the following instruments were used: BioShake iQ (QUANTIFOIL Instruments, Jena, Germany), Centrifuge 5424 (Eppendorf, Hamburg, Germany), UltiMate 3000 UHPLC System (Thermo Scientific, Germering, Germany) including a DAD detector (DAD-3000RS) coupled to an MSQ Plus single quadrupole mass spectrometer (Thermo Scientific, Austin, TX).Compounds 9−11, 15, LUZ5, and 17 were investigated with regard to their metabolism in vitro by incubation with mouse liver microsomes (MLMs) and analyses by HPLC-UV-MS according to an already published protocol. 81,82In brief, each of the test compounds (dissolved in DMSO, final concentration of 10 μM, final DMSO percentage of 1%) and MLMs (final protein concentration of 1 mg/mL) in PBS (pH 7.4) were preincubated in PBS (pH 7.4) for 5 min at 37 °C.Similarly preincubated NADPH (final concentration of 2 mM) was added to start the incubation.The final volume of each incubation mixture was 250 μL, and samples were prepared in duplicate.After the mixture had been gently shaken for 30 or 60 min at 37 °C, 1 mL of ice-cold CH 3 CN was added.The mixture was shaken vigorously for 30 s, rested on ice for 5 min, shaken for an additional 30 s, and then centrifuged at 14 000 rpm for 10 min.The supernatants were stored at 4 °C until they were measured by HPLC-UV-MS.As a positive control, samples with testosterone (final concentration of 20 μM) as the substrate were prepared like the test compounds, with or without NADPH and incubation for 60 or 90 min.Negative controls without NADPH as well as without both NADPH and MLM were also prepared for each test substance as well as controls without a substrate.
HPLC-UV-MS was performed on a Poroshell 120 EC-C18 column (100 mm × 3 mm, 2.7 μm) (Agilent Technologies, Waldbronn, Germany) at 25 °C (eluent A, H 2 O and 0.1% formic acid; eluent B, CH 3 CN and 0.1% formic acid) at a flow rate of 0.7 mL/min and monitoring a wavelength of 323 nm.The gradient elution method was as follows: 20% B from 0 to 1.5 min, 20% to 100% B from 1.5 to 10 min, 100% B from 10 to 12 min, and 20% B from 12 to 15 min.The MSQ Plus single quadrupole mass spectrometer was operated in positive and negative electrospray ionization mode: probe temperature of 550 °C, needle voltage of 3 V, and cone voltage of 75 V.On the basis of the obtained UV chromatograms, the percentage of unchanged compound was calculated from the peak area divided by the sum of peak areas of all relevant signals.The molar activity was determined using analytical radio-HPLC with a Reprosil-Pur C18-AQ column (250 mm × 4.6 mm, 5 μm) and 66% CH 3 CN/20 mM NH 4 OAc aq as the eluent at a flow rate of 1 mL/ min and UV detection at 312 nm.

Radiochemistry. Automated Radiosynthesis of [
Determination of Lipophilicity (logD 7.4 ).The logD 7.4 of [ 18 F]LUZ5-d 8 was experimentally determined in n-octanol/phosphate-buffered saline (PBS; 0.01 M, pH 7.4) at rt by the shake-flask method.The measurement was performed twice in triplicate. 22uantification of Radiometabolites.20 to 30 megabecquerels of [ 18 F]LUZ5-d 8 dissolved in ∼150 μL of isotonic saline was administered intravenously as a bolus in the tail vein of awake female CD-1 mice weighing ∼33 g (n = 3) and male Wistar rats weighing 240 and 380 g (n = 2).At 30 min p.i., the animals were anesthetized and blood was withdrawn by retrobulbar bleeding using glass capillaries.Immediately afterward, the animals were euthanized by cervical dislocation, and the released urine was sampled.Blood plasma was obtained from the whole blood sample by centrifugation (2 min, 8000 rpm, room temperature).In addition, the brain and spleen were isolated and homogenized in 1 mL of demineralized water on ice (1000 rpm, 10 strokes; glass vessel, PTFE plunger; Potter S, B. Braun Biotech International, Goettingen, Germany).
The samples were further processed for subsequent radiochromatographic analyses.Two consecutive extractions were performed as duplicates for plasma and brain determinations.Plasma and brain samples were added to an ice-cold mixture of either MeOH and H 2 O [9:1 (v/v); n = 3] or CH 3 CN and H 2 O [9:1 (v/v); n = 1] with extraction efficiencies of ≥94% for the former and ≥89% for the latter.The samples were vortexed for 3 min, incubated on ice for 5 min, vortexed for 3 min, and centrifuged at 10 000 rpm for 5 min.Supernatants were collected; the precipitates were redissolved in 100 μL of extraction solvent, and the extraction procedure was repeated.The activities of supernatants and precipitates were measured in a γcounter (1480 WIZARD, Fa.PerkinElmer), and the extraction efficiencies were calculated as the ratio of radioactivity in the supernatant to the radioactivity in the original sample (supernatant + precipitate).The supernatants from both extractions were combined, concentrated at 70 °C under argon up to a remaining volume of 100 μL, and subsequently analyzed by analytical radio-HPLC with a gradient system (see Quality Control).
PET Experiments.The in vivo biodistribution of [ 18 F]LUZ5-d 8 in male Wistar rats and a female CD-1 mouse was assessed by dynamic small animal PET (Nanoscan, Mediso, Budapest, Hungary), 30 min recordings, followed by T1-weighted (GRE, TR/TE = 15.0/2.4ms, 252/252, FA = 25°) magnetic resonance (MR) imaging with wholebody coils for anatomical correlation and attenuation correction.Animals were initially anesthetized with 5% isoflurane and placed on a thermostatically heated animal bed where anesthesia was maintained with 2% isoflurane in 60% oxygen/38% room air.The radiotracer was injected into the lateral tail vein (bolus within 5 s) at the start of the PET acquisition.
For the baseline study, 15 ± 3 MBq of the radiotracer was injected into three rats with a body weight of 230 ± 13 g.The blocking studies were conducted by the injection of 1.5 mg of the CB 2 R agonist GW405833 per kilogram (predissolved in 1:2:7 DMSO/Kolliphor/ 0.9% NaCl) 10 min prior to the radiotracer (21.6 ± 0.9 MBq) into three rats weighing 228 ± 10 g.Brain uptake studies were performed with two rats (body weights of 213 and 242 g, injected doses of 15.2 and 17.0 MBq, respectively); for the whole body biodistribution study in mouse, one animal was used (body weight of 30.6 g, injected dose of 15.2 MBq).
List-mode PET data were binned as a series of attenuationcorrected sinogram frames and reconstructed by ordered subset expectation maximization (OSEM3D) with four iterations, six subsets, and a voxel size of 0.4 mm 3 (Nucline version 2.01, Mediso).The analysis of reconstructed data sets was performed with PMOD version 4.103 (PMOD Technologies LLC, Zurich, Switzerland).Nonparametrical analysis of achieved time-activity curves (TACs) was performed with Microsoft Excel to determine the time to peak, the TAC peak value, and the area under the curve (AUC):

Figure 2 .
Figure 2. Common structural motifs for CB 2 R ligands, inspired by Spinelli et al.27

Figure 7 .
Figure 7. Molecular structures of 15 (left) and LUZ5 (right).For LUZ5, a disorder of the fluorobutyl substituent in two positions is observed (not shown).The carbon atom of the carborane cluster bearing the substituent could be identified, while the second carbon atom of the carborane unit was treated as homogeneously 5-fold disordered over the entire CB 4 ring.
9−11 and 15−17 were not soluble in aqueous solution without the addition of dimethyl sulfoxide (DMSO).Since a maximum of 1% DMSO can be used in the in vitro binding assay, stability measurements were performed in aqueous DMSO-d 6 to see especially if deboronation occurs in the presence of water.Approximately 10 mg of each compound (9−11 and 15−17) was dissolved in the deuterated NMR solvent and monitored via NMR spectroscopy in time intervals of a few minutes (after the addition of the solvent to the respective solid compound) to at least 23 days for the o-(9 and 15) and m-carborane derivatives (10 and LUZ5) and beyond one year for the pcarborane analogues (11 and 17) (Figures S68−S79).
, LUZ5, and 17) showed a stability beyond the binding affinity assay time frame with the following determined stability order: 9 < 15 < 10 < LUZ5 < 11 = 17.p-Carborane derivatives 11 and 17 were stable for more than one year.For compound LUZ5, additional stability tests were performed with an increased concentration of H 2 O or D 2 O in DMSO-d 6 [1:5 (v/v), in contrast to aqueous DMSO-d 6 for previous measurements].H 2 O or D 2 O was added to the clear solution, which immediately turned turbid indicating the formation of aggregates. 80However, investigation with 11 B{ 1 H} NMR spectroscopy was still possible and indicated that compound LUZ5 was stable in the solvent mixture with increased amounts of H 2 O for at least 19 days and in the mixture with increased amounts of D 2 O for at least 8 days (Figures S80 and S81).In Vitro Binding Assay.Target compounds 9−11, 15, LUZ5, and 17 were evaluated in a competitive binding affinity assay using hCB 2 R Chinese hamster ovary (CHO) cells and the agonistic high-affinity CB 2 R radioligand [ 3 H]WIN55212-2 (FigureS96).

Table 1 .
Binding Affinities of Target Compounds 9−11, 15, LUZ5, 17, and Reference Compounds to the CB 2 R a One value.b Mean of two values.c Mean of three values; σ is the standard deviation.

Figure 10 .
Figure 10.Dynamic PET studies of intravenously (i.v.) injected [ 18 F]LUZ5-d 8 in male Wistar rats.(A) Exemplary maximal intensity projections (MIPs) of magnetic resonance (MR) (T1-weighted), PET (averaged time frames as indicated), and merging of MR and PET of animals pretreated with or without 1.5 mg of GW405833/kg injected 10 min prior to the radiotracer (n = 3).(B) Time−activity curves (TACs) of the left ventricle and spleen in mean standardized uptake values (SUV mean ± standard deviation), as well as the spleen uptake normalized to blood [SUVr (spleen-toventricle)].(C) Horizontal sections of an exemplary PET recording of the head, with the dotted ellipse indicating the brain region.(D) Brain TACs of two different animals.

18
18mote-controlled radiosynthesis was performed using a Synchrom R&D EVO III automated synthesizer (Elysia-Raytest).Briefly, [18F]fluoride (5−12 GBq) was trapped on a Waters QMA cartridge, eluted with a H 2 O/CH 3 CN [1 mL, 1:4 (v/v)] solution containing 2.2.2-cryptand (K 2.2.2 ) (11 mg) and K 2 CO 3 (75 μL) into the reaction vessel, and dried via azeotropic distillation.To complete the azeotropic distillation, additional dry CH 3 CN (1.5 mL) was added.After complete dryness, precursor 19 (1 mg, 22.65 μmol) in CH 3 CN (1 mL) was added and the reaction mixture was stirred at 90 °C for 10 min.The reaction mixture was diluted with 4 mL of H 2 O/CH 3 CN (1:1), and the solution was transferred to the semipreparative HPLC instrument.[ 18 F]LUZ5-d 8 was collected via the HPLC collection vial containing 40 mL of H 2 O and trapped in the Sep-Pak C18 light cartridge.The cartridge was washed with 2 mL of H 2 O, and [ 18 F]LUZ5-d 8 was eluted with 1.3 mL of EtOH.This ethanolic solution was transferred outside of the shielded cell; the solvent was evaporated at 70 °C in a gentle stream of nitrogen for 5−10 min, and [ 18 F]LUZ5-d 8 was reconstituted in an isotonic saline solution for further biological characterization.The total synthesis time was ∼85 min.auto injector (100 μL sample loop), and a model UV-2070Plus detector coupled with a γ-detector (GABI Star; raytest Isotopenmessgeraẗe GmbH, Straubenhardt, Germany).Data analysis was performed with the Galaxie chromatography software (Agilent Technologies) using the chromatograms obtained at 254 nm.The radiochemical yield, radiochemical purity, and analyses of plasma and brain samples were assessed via reverse phase HPLC (RP-HPLC) in gradient mode (10% CH 3 CN/20 mM NH 4 OAc aq from 0 to 5 min, 10% → 90% CH 3 CN/20 mM NH 4 OAc aq from 5 to 18 min, 90% CH 3 CN/20 mM NH 4 OAc aq from 18 to 25 min, 90% → 10% CH 3 CN/20 mM NH 4 OAc aq from 25 to 26 min, and 10% CH 3 CN/20 mM NH 4 OAc aq from 26 to 30 min).